The reflex arc is a fundamental functional unit of the spinal cord. Disease or injury of any of the cellular elements in the unit can result in profound movement disorders. An engineering approach to this system would allow the evaluation of these problems and disorders in an in vitro system in a controlled, defined environment. This project proposes to develop a biologically integrated microfabricated silicon device to study synaptic communication development and reinnervation between motoneurons and myotubes. Our Hypothesis is that a combination of stem cells and growth factors will enable the reinnervation of muscle fibers and that human stem cells will innervate rat muscle in vitro to enable the in vivo evaluation of the above results. The motoneurons will be derived from embryonic rat as a control and compared/contrasted to adult rat motoneurons and motoneurons derived from human stem cells. The myotubes will be derived initially from embryonic rat, then adult rat and later adult human tissue. The primary goal of the project is to establish the ability of human stem cells to innervate adult rat muscle to determine the best conditions using the in vitro system to achieve in vivo reinnervation and functional recovery in a rat SCI model. Initially, in Aim 1 the motoneuron-to-muscle segment will be investigated at the single cell level to assess in vitro reinnervation in a system that is composed of patterned surfaces integrated with a MEMS construct. A chamber to mimic the PNS and CNS environment will be fabricated using MEMS fabrication methodology. Aim 1b examines the system created in Aim 1 where the target for the motoneurons is an adult myotube derived from human tissue. Aim 2 evaluates the system in the presence of growth factors. Aim 3 examines the effect of the presence of glial, Schwann and microglial cells on the functional capacity of the montoneuron to muscle segment. Aim 4 addresses the in vivo experiments that will evaluate the combination of growth factors and human stem cells optimized in Aims 1-3 to allow reinnervation of rat muscle and investigate functional recovery. An integration of fundamental neuroscience, cell biology, microsystem engineering, and surface chemistry will be implemented to build and test this hybrid device, leading eventually to designing schemes to prevent, diagnosis, and treat developmental abnormalities and chronic neurological/muscle disorders. New strategies for prosthetic and orthotic design and evaluation, and new approaches for spinal repair from the aspect of an activity dependent reinnervation of tissue are also envisioned. Finally, this technology if successful could be used to create functional assays to investigate human cells for maladies where animal models may not yet exist. The Nanoscience Technology Center at the University of Central Florida, the Neuroscience Institute at the Medical University of South Carolina, the Naval Research Laboratory (NRL), and Neuralstem (a biotech company devoted to human stem cell technology) have partnered to develop this technology.